by Milind C. Mahajan, and Sherman M. Weissman DNA-dependent adenosine triphosphatase (helicaselike transcription factor) activates β-globin transcription in K562 cells by Milind C. Mahajan, and Sherman M. Weissman Blood Volume 99(1):348-356 January 1, 2002 ©2002 by American Society of Hematology

Phylogenetically conserved sequences in the β-globin promoter and the HS2.(A) Conservation of the −112 to −140 bp region from the transcription start site of the adult human β-globin promoter. Phylogenetically conserved sequences in the β-globin promoter and the HS2.(A) Conservation of the −112 to −140 bp region from the transcription start site of the adult human β-globin promoter. The conserved sequences are underlined. The number at the left-hand side of the human sequence is the nucleotide number from the human globin locus (GenBank locus HUMHBB). (B) Comparison of the HS2 sequences at the NF-E2 region from mouse to human. The tandem NF-E2/AP1 sequences are double underlined. The numbers at the left-hand side of the sequences are the nucleotide numbers assigned to the respective globin locus. (C) Homology between the −112 to −139 bp β-promoter sequence (PCS) and the NF-E2 site (HS2-CS) of the HS2. The homologous sequences are underlined. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

Electrophoretic mobility shift assay (EMSA) to show in vitro sequence-specific binding of proteins to the PCS.The control lane displays EMSA bands obtained by incubating 5 μg nuclear extract with 2 ng 32P-labeled double-stranded PCS (oligo 1). Electrophoretic mobility shift assay (EMSA) to show in vitro sequence-specific binding of proteins to the PCS.The control lane displays EMSA bands obtained by incubating 5 μg nuclear extract with 2 ng 32P-labeled double-stranded PCS (oligo 1). In oligonucleotide competition assays, the32P-labeled double-stranded PCS of the β-promoter (oligo 1) was mixed with various amounts (molar excesses) of nonradioactive PCS-DNA as indicated at the top of the figure. A double-stranded oligonucleotide from −142 to −171 bp relative to the transcription start site of the β-promoter was used as a nonspecific (NS) oligonucleotide (oligo 25 and 26). The band between band 4 and 5 inconsistently appears on the gel. The EMSA bands are numbered and indicated by arrows. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

HLTF is present in several major DNA-protein complexes formed on the PCS.(A) Western blot of the K562 nuclear extract with HLTF antibody A and B shows a prominent band of 115 kd. HLTF is present in several major DNA-protein complexes formed on the PCS.(A) Western blot of the K562 nuclear extract with HLTF antibody A and B shows a prominent band of 115 kd. A minor faint band below may be the degradation product. K562 nuclear extract (50 μg) was resolved on a 12% SDS-PAGE. The HLTF antibodies are indicated on the top, and the molecular weight markers are indicated at the left-hand side. (B) EMSA with 32P-labeled PCS oligonucleotide (oligo 1) and K562 nuclear extracts in the presence (+) and absence (−) of HLTF antibodies A and B. Normal rabbit serum and antibodies against p18 Maf and Sin3A served as control. In a standard 20-μL EMSA reaction mixture, 2 μL each of the antibody mentioned at the top of the panel and 5 μg nuclear extracts were preincubated for 10 minutes before the addition of the 32P-labeled DNA probe. The binding mix was incubated for an additional 10 minutes at room temperature after the addition of the DNA probe and analyzed on a 5% polyacrylamide gel. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

Presence of HLTF in some of the DNA-protein complexes formed on the HS2-CS.(A) In vitro sequence-specific binding of K562 nuclear proteins to the HS2-CS. Presence of HLTF in some of the DNA-protein complexes formed on the HS2-CS.(A) In vitro sequence-specific binding of K562 nuclear proteins to the HS2-CS. The DNA probe used in the EMSA is 32P-labeled double-stranded HS2-CS sequence (oligo 10). The control lane is EMSA without nonradioactive HS2-CS oligo. The molar excesses of nonradioactive DNA probe used in the competitive EMSA are shown on the top of the lanes. The NS oligo is described in Figure 2. The EMSA bands are numbered and indicated by arrow marks. Free probe lane displays EMSA pattern in the absence of K562 nuclear extracts. (B) HLTF is present in 2 EMSA bands obtained with a double-stranded HS2-CS sequence and K562 nuclear extracts. Gel supershift/neutralization assay with K562 nuclear extract and 32P-labeled HS2-CS oligonucleotide in the presence (+) and absence (−) of HLTF antibody A and B, normal rabbit serum, and anti-Sin3A antibody. The assay conditions were the same as described in Figure 3. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

Mutations in the PCS affect EMSA bands and β-promoter transcription Mutations in the PCS affect EMSA bands and β-promoter transcription.(A) Mutations in the PCS influence the DNA-protein complex formation. Mutations in the PCS affect EMSA bands and β-promoter transcription.(A) Mutations in the PCS influence the DNA-protein complex formation. EMSA was carried out with the wild-type PCS and its mutant variants by using K562 nuclear extracts. The 20-μL reaction mixture contained 2 ng 32P-labeled double-stranded DNA (∼100 000 cpm) in the binding buffer and 5 μg nuclear extract. The sequences of the sense strand of each of the double-stranded 32P-labeled DNA probes is as follows: wild type, oligo 1; AT to CC substitution, oligo 2; Δ GACAGGTA, oligo 3; and Δ CGGCT, oligo 4 (Table 1). Sequences from the pGL3 plasmid-cloning sites were included in oligo 3 and 4 to maintain their length equivalent to the wild-type PCS oligonucleotide. The free probe lane is the mixture of all the above sequences in the binding reaction mixture without nuclear extracts. The EMSA bands are numbered and indicated by arrows. (B) Functional effects of mutations in the PCS. The wild-type β-promoter from −136 to +54 bp and its mutants were generated by PCR and cloned into the Hind III-XhoI site of the pGL3. The forward primers used in the PCR are as follows: wild-type β-promoter, oligo 5; β-promoter with AT to CC substitution in the PCS, oligo 6; β-promoter with Δ GACAGGTA in the PCS, oligo 7; and β-promoter with Δ CGGCT in the PCS, oligo 8. Oligo 9 served as the reverse primer in all the PCR reactions. A 736-bp HS2 (from nucleotides 8486 to 9222 on the human β-globin locus) was cloned at the BamHI-SalI site of the pGL3. All the values in the bar diagram are the averages of 3 to 8 separate experiments, most of which were done in duplicate. The luciferase activities are normalized as activity per 10 μg total protein. Horizontal lines above each bar indicate 1 SD in the average of the experimental values. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

Binding of the recombinant 38-kd HLTF to wild-type and mutated PCS Binding of the recombinant 38-kd HLTF to wild-type and mutated PCS.A cDNA clone coding for the N-terminal 38-kd HLTF (aa 2 to aa 332) isolated by screening the λ gt11 cDNA library of K562 cells was cloned into pTrcHis2TOPO vector and expressed in E colicells. Binding of the recombinant 38-kd HLTF to wild-type and mutated PCS.A cDNA clone coding for the N-terminal 38-kd HLTF (aa 2 to aa 332) isolated by screening the λ gt11 cDNA library of K562 cells was cloned into pTrcHis2TOPO vector and expressed in E colicells. The protein extracts of uninduced and IPTG-induced bacterial cells were used in the EMSA. The wild-type and mutant versions of the PCS oligonucleotides are indicated, and their sequences are described in Figure 5. The 20-μL EMSA binding mixture contained 2 ng32P-labeled double-stranded DNA (∼100 000 cpm) in the binding buffer and 1 μL E coli protein extract. The binding reaction was started by the addition of the32P-labeled DNA probe. The reaction mixture was incubated for 10 minutes at room temperature and then analyzed on a 5% polyacrylamide gel. For gel supershift/neutralization assay with antibody A, the bacterial extract, and the antibody were incubated for 5 minutes before the addition of the 32P DNA probe. The EMSA band obtained with 38-kd HLTF is shown by an arrow. The upper EMSA band is due to a bacterial protein in the extract. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

Overexpression of HLTF in K562 cells and its effect on the β-globin transcription.(A) Western blot of total protein extract of K562 cells stably transfected with pCIneo vector alone and pCIneo containing HLTF with poly His tag at its N-terminal end. Overexpression of HLTF in K562 cells and its effect on the β-globin transcription.(A) Western blot of total protein extract of K562 cells stably transfected with pCIneo vector alone and pCIneo containing HLTF with poly His tag at its N-terminal end. Protein (70 μg) was loaded in each lane. The cell-free extracts are indicated at the top of the panel, and standard molecular weight marker positions are indicated at the left-hand side. The arrows mark points at the HLTF band. The antibodies against HLTF and poly His used to probe the blot are mentioned at the bottom of the panel. In the blot probed with the anti-His antibody, multiple bands found below the 115-kd HLTF band are common to both the lanes. These bands presumably are due to the binding of anti poly-His antibody to proteins containing short stretches of histidines. (B) Transient transfections of −136 to +54 bp β-promoter (cloned into theHindIII-XhoI site of the pGL3) into normal and HLTF-overexpressing cells. (C) Transient transfection of pGL3 constructs containing β-promoter and its mutant variants and HS2 at the enhancer site. These constructs are described in Figure 5. The values in the bar diagram are the averages of 12 experiments, including 3 separate experiments performed in duplicate with each of the 2 separate pools of K562 cells stably transfected with HLTF. The luciferase activities are normalized as activity per 10 μg total protein. Horizontal lines above each bar indicate 1 SD in the average of the experimental values. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology

RT-PCR of total RNA from normal and HLTF-overexpressing K562 cells RT-PCR of total RNA from normal and HLTF-overexpressing K562 cells.RT-PCR conditions are described in the “Materials and methods” section. RT-PCR of total RNA from normal and HLTF-overexpressing K562 cells.RT-PCR conditions are described in the “Materials and methods” section. After 18 (left) or 22 (right) cycles, the PCR products were run on a 6% polyacrylamide gel for 2.5 hours, dried, and exposed to x-ray film. The data are representative of 3 independent experiments. K562 + HLTF transfection-1 and transfection-2 are the 2 pools of K562 cells stably transfected with pCIneo containing HLTF in 2 independent transfections. The exposure times were adjusted for each set of PCR to obtain bands of optimum intensity. The autoradiogram exposure times are as follows: 16 hours at room temperature (RT) for β-globin, 12 hours at −80°C for EKLF, 5 hours at RT for ε-globin, 15 minutes at RT for γ-globin, 1 hour at RT for β-actin, and 0.5 hours at RT for GAPDH. Milind C. Mahajan, and Sherman M. Weissman Blood 2002;99:348-356 ©2002 by American Society of Hematology